CN110885880A - Primer, method and kit for human mitochondria whole genome detection - Google Patents
Primer, method and kit for human mitochondria whole genome detection Download PDFInfo
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Abstract
The invention discloses a primer, a method and a kit for human mitochondria whole genome detection. The primers comprise forward and reverse primers for amplifying and covering 37 gene coding regions of a mitochondria whole genome (16569bp), and the base sequences of the primers are shown as SEQ ID NO: 3-222. The detection method based on the NGS technology comprises the following steps: combining a specific primer with a target template DNA sequence, amplifying a target region of a sample to be detected by using a universal primer, purifying a library by using magnetic beads, carrying out high-throughput sequencing on the obtained library and analyzing the mutation of mitochondrial genes. The primer, the method and the kit disclosed by the invention can be used for accurately detecting rare mutation of mitochondrial DNA, realize high cost performance, minimum manual operation and ultrahigh sensitivity, provide guidance for selective medication of familial mitochondrial patients, provide guidance for genetic susceptibility, assist in diagnosing mitochondrial diseases caused by mitochondrial DNA mutation and effectively reduce morbidity risk.
Description
Technical Field
The invention relates to the field of gene mutation detection of biotechnology, and in particular relates to a primer, a method and a kit for human mitochondrial whole genome detection.
Background
Mitochondria (mitochondria) are the main sites and energy supply centers for the oxidation of cellular material. Can provide more than 90 percent of Adenosine Triphosphate (ATP) required by metabolism, regulate apoptosis and are the main sites for generating free Radicals (ROS) in cells. Mitochondria contain DNA molecules, called human chromosome 25, which are organelles outside the nucleus containing genetic information and expression systems, and are characterized by non-mendelian inheritance, also called extranuclear inheritance. Mitochondrial diseases (mitochondriopaths) are a group of multisystem or tissue-specific diseases that arise from genetic defects that cause defects in mitochondrial metabolic enzymes, resulting in ATP synthesis impairment, and insufficient energy sources.
The human mitochondrial genome is 16569bp in length and contains 37 genes in total, 13 of which encode functional proteins (respiratory chain complex), 2 of which encode rRNA (16S and 12S), and 2 of which encode tRNA. The incidence of mitochondrial diseases is reported in the literature to be about 1/5000, mitochondrial diseases can be caused by nuclear genomic DNA mutations or mitochondrial DNA mutations, and mitochondrial diseases caused by mitochondrial DNA mutations account for about 15%. The common mitochondrial diseases mainly comprise mitochondrial diseases with hyperlactacidosis and stroke-like attack syndrome (MELAS), myoclonic epilepsy with fleshy-like red fiber syndrome (MERRF), chronic progressive external ophthalmoplegia syndrome (KSS), primary mitochondrial encephalopathy Leigh syndrome, Leber Hereditary Optic Neuropathy (LHON), Pearson syndrome and the like. Mitochondrial DNA mutations are very common in the human population and it has been reported in the literature that one of 200 newborns carries one of 10 common mitochondrial DNA pathogenicity mutations. Mitochondrial DNA mutation is distributed throughout the whole mitochondrial genome, more than 500 kinds of point mutation can be found, and the common point mutation sites are m.3243A > G, m.3460G > A, m.1555A > G, m.8344A > G and the like.
Currently, Sanger sequencing is a commonly used mitochondrial gene mutation detection method, but Sanger sequencing has some defects: the Sanger sequencing method can only detect the variation with the mutation rate of more than 20 percent generally, and has poor sensitivity; for detecting all mitochondrial gene coding sequences, the period is long, the cost is high, the operation is complex, the detection flux is low, accurate data is difficult to obtain through one-time sequencing, false positive can be avoided only by repeating the sequencing for many times, and the requirement of practical application is difficult to meet. In addition, the method of separating mitochondria by an ultracentrifuge and extracting DNA requires a large tissue sample, and the early experimental process is complicated.
Based on the reasons, the invention relates to a method and a kit for designing a set of novel multiplex PCR primers and simultaneously combining a second-generation sequencing technology to develop sequencing and analysis aiming at mitochondrial gene mutation.
Disclosure of Invention
The invention aims to provide a primer, a method and a kit for detecting a mutation site of a human mitochondrial whole gene coding sequence based on multiplex PCR and high-throughput sequencing technologies, and aims to solve the problems of high cost, multiple steps, low sensitivity, long period, easy pollution and the like in the prior art.
In one aspect of the invention, a primer for detecting a whole genome of a mitochondria is provided, wherein the sequence of the universal primer is shown as SEQ ID NO.1 and SEQ ID NO. 2;
the specific primer sequence for amplifying the whole genome of mitochondria is shown as SEQ ID NO. 3-222;
in another aspect of the present invention, a kit for detecting a whole genome of mitochondria is provided, which comprises the following main components:
another aspect of the present invention provides a multiplex PCR system for detecting a mitochondrial whole genome, which comprises:
| components | Volume of |
| 2 × PCR mixture | 6.25μL |
| Specific primer | 0.5μL |
| Universal primer R5 | 0.5μL |
| Universal primer R7 | 0.5μL |
| DNA template, 50ng | xμL |
| Nuclease-free water | 4.75-xμL |
| Total volume | 12.5μL |
In another aspect of the present invention, there is provided a method for detecting a mitochondrial whole genome, comprising the steps of:
A. preparing a DNA template.
B. Amplifying a target region of a genome of a sample to be detected by a multiplex PCR method:
1) sterile, nuclease-free 200. mu.L PCR tubes were prepared and placed on ice.
2) Preparing a multiplex PCR system, lightly blowing and uniformly mixing, centrifuging and placing the tube on ice.
3) The tube was placed in a PCR instrument and run according to the following program:
| step (ii) of | Temperature (. degree.C.) | Time of day | |
| Activation of | 1 | 98 | 5min |
| Denaturation of the |
2 | 98 | 15sec |
| Annealing |
3 | 65 | 120min |
| Extension of | 4 | 68 | 60sec |
| Extension of | 5 | 72 | 60sec |
| Returning to the |
6 | ||
| Denaturation of the material | 7 | 98 | 15sec |
| Annealing universal primer | 8 | 65 | 30sec |
| Extension of | 9 | 68 | 30sec |
| Extension of | 10 | 72 | 30sec |
| Returning to the step 7 for 1 cycle | 11 | ||
| Denaturation of the material | 12 | 98 | 15sec |
| Extension of | 13 | 68 | 60sec |
| Extension of | 14 | 72 | 60sec |
| Returning to the step 12 to circulate for 25 times | 15 | ||
| Extension of | 16 | 72 | 10min |
| Holding | 17 | 4 | Holding |
C. The library was purified using magnetic beads.
D. The resulting library was subjected to high throughput sequencing and mitochondrial whole genome mutation information was analyzed.
The invention has the beneficial effects that: the invention is based on a high-throughput sequencing platform, adopts specific primers and a multiplex PCR technology, and can quickly and accurately realize the detection of mitochondrial gene mutation. The invention comprises the following steps: (1) the novel primer design is provided, the mitochondrial gene coding region is designed according to 100% amplification coverage, the formation of primer dimer is greatly reduced, and meanwhile, a longer real sequence is contained in an amplicon, so that the primer specificity is fundamentally improved, and the amplification efficiency is improved; (2) using a novel multiplex PCR technique, the control specific primers only function in two cycles of initial target capture, and then automatically switch to library amplification with the same efficiency, which is guided by the universal primers, to obtain a very uniform library; (3) the method is simple to operate, can finish the preparation work of the targeted library of the mitochondrial gene in one step by manual operation within 5 minutes, saves the cost and has wide clinical application range; (4) the whole process has no extra process of opening the pipe, thereby avoiding the pollution risk brought by the manpower.
Drawings
In order to make the purpose, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings:
FIG. 1: genome DNA quality inspection chart with different degradation degrees
FIG. 2: quality inspection chart of library products after specific purification
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The following examples are presented to further illustrate the invention but should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1
The performance of the kit was verified by using plasmid DNA with known site mutations to mimic a series of original templates with different mutation frequencies.
A. Template preparation for different mutation ratios
Plasmid DNA mutated at known sites (chrMT:11778G > A, GRCh37.p13) was used in a ratio mix with wild type DNA to give the original template with mutation frequencies of 0.05%, 1%, 2%, 5%, 10%, respectively.
B. Multiplex PCR method for amplifying target region of sample genome
1) Preparing a sterile, nuclease-free 200 μ L PCR tube, and placing on ice; PCR reaction systems were prepared as follows, with 10ng template per reaction, taking care to avoid cross-contamination.
| Components | Volume of |
| 2 × PCR mixture | 6.25μL |
| Specific primer | 0.5μL |
| Universal primer R5 | 0.5μL |
| Universal primer R7 | 0.5μL |
| DNA template, 50ng | 1μL |
| Nuclease-free water | 3.75μL |
| Total volume | 12.5μL |
2) After preparation, vortex mixing, centrifuge separation, PCR reaction tube on ice.
3) Placing the PCR reaction tube in a nucleic acid amplification instrument, and operating according to the following reaction program:
| step (ii) of | Temperature (. degree.C.) | Time of day | |
| Activation of | 1 | 98 | 5min |
| Denaturation of the |
2 | 98 | 15sec |
| Annealing |
3 | 65 | 120min |
| Extension of | 4 | 68 | 60sec |
| Extension of | 5 | 72 | 60sec |
| Returning to the |
6 | ||
| Denaturation of the material | 7 | 98 | 15sec |
| Annealing universal primer | 8 | 65 | 30sec |
| Extension of | 9 | 68 | 30sec |
| Extension of | 10 | 72 | 30sec |
| Returning to the step 7 for 1 cycle | 11 | ||
| Denaturation of the material | 12 | 98 | 15sec |
| Extension of | 13 | 68 | 60sec |
| Extension of | 14 | 72 | 60sec |
| Returning to the step 12 to circulate for 25 times | 15 | ||
| Extension of | 16 | 72 | 10min |
| Holding | 17 | 4 | Holding |
C. Purification of libraries using magnetic beads
1) The purified magnetic beads were removed from 2-8 ℃, equilibrated at room temperature for 20 minutes, and then blown up and mixed well.
2) Supplementing nuclease-free water into the PCR product to 80 mu L, transferring the PCR product to a new nuclease-free centrifuge tube, adding 68 mu L of uniformly mixed magnetic beads, and performing air-assisted beating for 10 times to fully mix the DNA and the magnetic beads.
3) Incubate for 5 minutes at room temperature.
4) The centrifuge tubes were placed on a magnetic rack for 5 minutes at room temperature until the solution was clear.
5) Transfer the supernatant to a new nuclease-free centrifuge tube without disturbing the magnetic beads.
6) Adding 12 mu L of the well-mixed magnetic beads into the centrifuge tube in the step 5, and blowing and beating the mixture for 10 times by using a 200 mu L pipette gun to mix the mixture evenly.
7) Incubate for 5 minutes at room temperature.
8) The centrifuge tubes were placed on a magnetic rack for 5 minutes at room temperature until the solution was clear.
9) Discard the supernatant without disturbing the magnetic beads.
10) Keep the centrifuge tube on the magnetic rack and add 200 μ L each of freshly prepared 80% ethanol, taking care not to disturb the magnetic beads.
11) Carefully aspirate the supernatant after at least 30 seconds of standing.
12) Repeat steps 10-11. Ethanol cleaning is carried out for two times in total, and no ethanol residue is ensured.
13) The centrifuge tubes were removed from the magnetic rack and allowed to dry at room temperature for 5-10 minutes, taking care not to overdry.
14) Resuspend the magnetic beads with 20. mu.L of 10mM Tris-HCl buffer (pH 8.0). Gently blow and mix the suspension while resuspending. After 5 minutes at room temperature, the centrifuge tubes were placed on a magnetic rack for 5 minutes at room temperature until the solution was clear. Transfer supernatant to a new nuclease-free centrifuge tube without disturbing the magnetic beads.
15) Adding 20 mu L of the well-mixed magnetic beads into the centrifuge tube in the step 14, and blowing and beating for 10 times to mix uniformly.
16) Repeating steps 7 to 13. The magnetic beads were resuspended with 10. mu.L of 10mM Tris-HCl buffer, pH 8.0. Gently blow and mix the suspension while resuspending. After 5 minutes at room temperature, the centrifuge tubes were placed on a magnetic rack for 5 minutes at room temperature until the solution was clear. Transfer supernatant to a new nuclease-free tube without disturbing the beads.
17) Specific purified library Using Agilent
The 2100 instrument carries out quantification and quality inspection, and the obtained high-throughput library is subjected to 150bp double-end sequencing of the illumina Miseq platform, and the data volume requires 0.03G base pair.
D. Analysis of results
1) Analysis of data
And (3) carrying out data quality analysis on the data after the sequencing is off the machine, removing invalid data, comparing a human genome (hg19) to enable reads to correspond to corresponding genome positions, and comparing through software to obtain mutation types and mutation frequencies.
2) Comparison of results
The results after sequencing analysis are shown in the following table in relation to the theoretical mutation frequency.
TABLE alignment of measured mutation frequencies to theoretical mutation frequencies
The result shows that the actually measured mutation frequency of the kit on the known site is similar to the preset mutation frequency. The kit is proved to be high in specificity and accuracy.
Example 2
And (3) using the genomic DNA with different degradation degrees as a template, analyzing the correlation among sequencing results, and verifying the performance of the kit.
A. Preparation of genomic DNA with varying degrees of degradation
250ng of Hela genomic DNA (NEB, N4006S) was used as a raw material
The dsDNAfragmentase (NEB, M0348S) is used for digesting the raw material at 37 deg.C for 0, 10, 20, 30, 50min, etc., and the digested product is Agilent
The 2100 instrument performs quantification and quality control. As shown in FIG. 1, the raw material was significantly degraded after digestion at 37 ℃ for 20min, and the degradation was more severe the longer the treatment time.
B. Multiplex PCR method for amplifying target region of sample genome
1) Preparing a sterile, nuclease-free 200 μ L PCR tube, and placing on ice; PCR reaction systems were prepared as follows, with 10ng template per reaction, taking care to avoid cross-contamination.
| Components | Volume of |
| 2 × PCR mixture | 6.25μL |
| Specific primer | 0.5μL |
| Universal primer R5 | 0.5μL |
| Universal primer R7 | 0.5μL |
| DNA template, 10 ng/. mu.L | 1μL |
| Nuclease-free water | 3.75μL |
| Total volume | 12.5μL |
2) After preparation, vortex mixing, centrifuge separation, PCR reaction tube on ice.
3) Placing the PCR reaction tube in a nucleic acid amplification instrument, and operating according to the following reaction program:
C. purification of libraries using magnetic beads
1) The purified magnetic beads were removed from 2-8 ℃, equilibrated at room temperature for 20 minutes, and then blown up and mixed well.
2) Supplementing nuclease-free water into the PCR product to 80 mu L, transferring the PCR product to a new nuclease-free centrifuge tube, adding 68 mu L of uniformly mixed magnetic beads, and performing air-assisted beating for 10 times to fully mix the DNA and the magnetic beads.
3) Incubate for 5 minutes at room temperature.
4) Place the centrifuge tube on a magnetic rack for 5 minutes until the solution is clear.
5) Transfer the supernatant to a new nuclease-free centrifuge tube without disturbing the magnetic beads.
6) Adding 12 mu L of the well-mixed magnetic beads into the centrifuge tube in the step 5, and blowing and beating the mixture for 10 times by using a 200 mu L pipette gun to mix the mixture evenly.
7) Incubate for 5 minutes at room temperature.
8) The centrifuge tubes were placed on a magnetic rack for 5 minutes at room temperature until the solution was clear.
9) Discard the supernatant without disturbing the magnetic beads.
10) Keep the centrifuge tube on the magnetic rack and add 200 μ L each of freshly prepared 80% ethanol, taking care not to disturb the magnetic beads.
11) Carefully aspirate the supernatant after at least 30 seconds of standing.
12) Repeat steps 10-11. Ethanol cleaning is carried out for two times in total, and no ethanol residue is ensured.
13) The centrifuge tubes were removed from the magnetic rack and allowed to dry at room temperature for 5-10 minutes, taking care not to overdry.
14) Resuspend the magnetic beads with 20. mu.L of 10mM Tris-HCl buffer (pH 8.0). Gently blow and mix the suspension while resuspending. After 5 minutes at room temperature, the centrifuge tubes were placed on a magnetic rack for 5 minutes at room temperature until the solution was clear. Transfer supernatant to a new nuclease-free centrifuge tube without disturbing the magnetic beads.
15) Adding 20 mu L of the well-mixed magnetic beads into the centrifuge tube in the step 14, and blowing and beating for 10 times to mix uniformly.
16) Repeating steps 7 to 13. The magnetic beads were resuspended with 10. mu.L of 10mM Tris-HCl buffer, pH 8.0. Gently blow and mix the suspension while resuspending. After 5 minutes at room temperature, the centrifuge tubes were placed on a magnetic rack for 5 minutes at room temperature until the solution was clear. Transfer supernatant to a new nuclease-free tube without disturbing the beads.
17) Specific purified library Using Agilent
The 2100 instrument performed quantitation and quality control, and the results are shown in FIG. 2 (only Tube1 amplification product is exemplified): the length of the target product is concentrated around 330 bp; except for the template DNA degraded to about 200bp (treated at 37 ℃ for 50min), no non-specific product is generated in the rest templates after PCR.
18) The resulting high throughput library was subjected to illumina Miseq platform 150bp paired-end sequencing, with data size requirements of 0.03G base pairs.
D. Analysis of results
1) Analysis of data
And (3) carrying out data quality analysis on the data after the sequencing is off the machine, removing invalid data, comparing a human genome (hg19) to enable reads to correspond to corresponding genome positions, and comparing through software to obtain mutation types and mutation frequencies.
6. Analysis of results
And (3) carrying out correlation analysis on the sequencing data of the amplified products of the DNAs with different degradation degrees, wherein the analysis results are shown in the following table:
table correlation coefficient analysis
The results show that the kit can tolerate the degradation of the template DNA to different degrees, and the correlation between the sequencing result and the complete genome is 0.808 at least when the complete genome DNA is digested for 50 min. The results can prove the stability of the performance of the kit.
Sequence listing
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<222>(22)..(37)
<223>n is a, c, g, or t
<400>25
ccttggcacc cgagaattcc annnnnnnnn nnnnnnnact tggacgaacc ag 52
<210>26
<211>56
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>26
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnntgcgc caggtt 56
<210>27
<211>59
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>27
ccttggcacc cgagaattccannnnnnnnn nnnnnnnacc caaataaagt ataggcgat 59
<210>28
<211>62
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>28
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnnttctt aggtagctcg 60
tc 62
<210>29
<211>50
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>29
ccttggcacc cgagaattcc annnnnnnnn nnnnnnngct aagacccccg 50
<210>30
<211>65
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>30
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnntctac aaggtttttt 60
cctag 65
<210>31
<211>58
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>31
ccttggcacc cgagaattcc annnnnnnnn nnnnnnntct aaatcccctt gtaaattt 58
<210>32
<211>63
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>32
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnngtgat agattggtcc 60
aat 63
<210>33
<211>63
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>33
ccttggcacc cgagaattcc annnnnnnnn nnnnnnntcc caaacatata actgaactcc 60
tca 63
<210>34
<211>58
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>34
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnngcctg tgttgggt 58
<210>35
<211>58
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>35
ccttggcacc cgagaattcc annnnnnnnn nnnnnnnacc aacaagtcat tattaccc 58
<210>36
<211>54
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>36
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnnaccgc ggcc 54
<210>37
<211>54
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>37
ccttggcacc cgagaattcc annnnnnnnn nnnnnnnacc agtattagag gcac 54
<210>38
<211>60
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>38
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnntgact ggtgaagtct 60
<210>39
<211>50
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>39
ccttggcacc cgagaattcc annnnnnnnn nnnnnnngga gcagaaccca 50
<210>40
<211>63
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>40
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnntcctg atccaacatc 60
gag 63
<210>41
<211>57
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>41
ccttggcacc cgagaattcc annnnnnnnn nnnnnnntcc atatcaacaa tagggtt 57
<210>42
<211>60
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>42
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnntcgta cagggaggaa 60
<210>43
<211>49
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>43
ccttggcacc cgagaattcc annnnnnnnn nnnnnnntcc aggtcggtt 49
<210>44
<211>58
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>44
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnnttggc catgggta 58
<210>45
<211>48
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>45
ccttggcacc cgagaattcc annnnnnnnn nnnnnnngca gagcccgg 48
<210>46
<211>57
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>46
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnnggcgt cagcgaa 57
<210>47
<211>49
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>47
ccttggcacc cgagaattcc annnnnnnnn nnnnnnngcc ccaacgttg 49
<210>48
<211>60
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>48
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnngccta ggttgaggtt 60
<210>49
<211>56
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>49
ccttggcacc cgagaattcc annnnnnnnn nnnnnnnacc cccctcccca taccca 56
<210>50
<211>59
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>50
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnntgatg gctagggtg 59
<210>51
<211>49
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>51
ccttggcacc cgagaattcc annnnnnnnn nnnnnnngca ctgcgagca 49
<210>52
<211>63
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>52
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnngcctg agactagttc 60
gga 63
<210>53
<211>53
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>53
ccttggcacc cgagaattcc annnnnnnnn nnnnnnngca gagaccaacc gaa 53
<210>54
<211>56
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>54
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnngggga gagtgc 56
<210>55
<211>56
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>55
ccttggcacc cgagaattcc annnnnnnnn nnnnnnnacc ctcaccacta caatct 56
<210>56
<211>59
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>56
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnngctag ggtgagtgg 59
<210>57
<211>55
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>57
ccttggcacc cgagaattcc annnnnnnnn nnnnnnnacc aactcataca cctcc 55
<210>58
<211>58
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>58
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnnggatg gggtgtga 58
<210>59
<211>60
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>59
ccttggcacc cgagaattcc annnnnnnnn nnnnnnnttt ctaggactat gagaatcgaa 60
<210>60
<211>60
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>60
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnntgtga tgagtgtgcc 60
<210>61
<211>49
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>61
ccttggcacc cgagaattcc annnnnnnnn nnnnnnngcc caacccgtc 49
<210>62
<211>56
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>62
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnntgcgg ttgctt 56
<210>63
<211>50
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>63
ccttggcacc cgagaattcc annnnnnnnn nnnnnnnaca gaagctgcca 50
<210>64
<211>57
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>64
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnnggtgc cttgggt 57
<210>65
<211>52
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>65
ccttggcacc cgagaattcc annnnnnnnn nnnnnnnttc tgagtcccag ag 52
<210>66
<211>59
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>66
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnnacctc aactgcctg 59
<210>67
<211>53
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>67
ccttggcacc cgagaattcc annnnnnnnn nnnnnnngcc ttctcctcac tct 53
<210>68
<211>62
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>68
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnngtagt aggaatgcgg 60
ta 62
<210>69
<211>55
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>69
ccttggcacc cgagaattcc annnnnnnnn nnnnnnnacc ctaacataac cattc 55
<210>70
<211>67
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>70
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnntgttt tttgtgaatt 60
cttcgat 67
<210>71
<211>48
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>71
ccttggcacc cgagaattcc annnnnnnnn nnnnnnngcc tgcccccg 48
<210>72
<211>63
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>72
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnnggtgg gttttgtatg 60
ttc 63
<210>73
<211>57
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>73
ccttggcacc cgagaattcc annnnnnnnn nnnnnnntcc ccatatctaa caacgta 57
<210>74
<211>60
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>74
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnnactta ctgagggctt 60
<210>75
<211>51
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>75
ccttggcacc cgagaattcc annnnnnnnn nnnnnnnaca gaccaagagc c 51
<210>76
<211>65
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>76
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnntgtta actaagtgtt 60
tgtgg 65
<210>77
<211>58
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>77
ccttggcacc cgagaattcc annnnnnnnn nnnnnnngcc cttactagac caatggga 58
<210>78
<211>63
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>78
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnngcctc tttttaccag 60
ctc 63
<210>79
<211>63
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>79
ccttggcacc cgagaattcc annnnnnnnn nnnnnnngaa tttgcaattc aatatgaaaa 60
tca 63
<210>80
<211>69
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>80
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnngaata ataggtatag 60
tgttccaat 69
<210>81
<211>59
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>81
ccttggcacc cgagaattcc annnnnnnnn nnnnnnngtt gactattctc tacaaacca 59
<210>82
<211>57
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>82
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnngggct gtgacga 57
<210>83
<211>53
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>83
ccttggcacc cgagaattcc annnnnnnnn nnnnnnnacc acatctacaa cgt 53
<210>84
<211>64
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>84
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnntccac tatagcagat 60
gcga 64
<210>85
<211>55
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>85
ccttggcacc cgagaattcc annnnnnnnn nnnnnnntcc ctctctccta ctcct 55
<210>86
<211>70
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>86
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnnggttt tatattgata 60
attgttgtga 70
<210>87
<211>56
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>87
ccttggcacc cgagaattcc annnnnnnnn nnnnnnntgt ctcctctatc ttaggg 56
<210>88
<211>63
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>88
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnnttggt atagaatggg 60
gtc 63
<210>89
<211>52
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>89
ccttggcacc cgagaattcc annnnnnnnn nnnnnnnacc ttcttcgacc cc 52
<210>90
<211>65
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>90
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnngccaa ttgatatcat 60
agctc 65
<210>91
<211>56
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>91
ccttggcacc cgagaattcc annnnnnnnn nnnnnnntcc ggaaaaaaag aaccat 56
<210>92
<211>58
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>92
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnntggag tgtggcga 58
<210>93
<211>49
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>93
ccttggcacc cgagaattcc annnnnnnnn nnnnnnntcc ccaccggcg 49
<210>94
<211>63
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>94
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnnttgat aggacatagt 60
gga 63
<210>95
<211>53
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>95
ccttggcacc cgagaattcc annnnnnnnn nnnnnnntcg tactacacga cac 53
<210>96
<211>63
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>96
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnngccga gaaagtgttg 60
tgg 63
<210>97
<211>54
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>97
ccttggcacc cgagaattcc annnnnnnnn nnnnnnntca tattcatcgg cgta 54
<210>98
<211>66
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>98
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnnggttc ttctactatt 60
aggact 66
<210>99
<211>52
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>99
ccttggcacc cgagaattcc annnnnnnnn nnnnnnntga tttgagaagc ct 52
<210>100
<211>59
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>100
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnnaccta cttgcgctg 59
<210>101
<211>61
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>101
ccttggcacc cgagaattcc annnnnnnnn nnnnnnnttt gtcaaagtta aattataggc 60
t 61
<210>102
<211>69
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>102
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnntgaga tgttagtatt 60
agttagttt 69
<210>103
<211>55
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>103
ccttggcacc cgagaattcc annnnnnnnn nnnnnnngcc cttttcctaa cactc 55
<210>104
<211>63
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>104
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnngccaa ttgatttgat 60
ggt 63
<210>105
<211>57
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>105
ccttggcacc cgagaattcc annnnnnnnn nnnnnnntcc tttacataac agacgag 57
<210>106
<211>67
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>106
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnntgtaa ttattatacg 60
aatgggg 67
<210>107
<211>53
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>107
ccttggcacc cgagaattcc annnnnnnnn nnnnnnntcc ttgacgttga caa 53
<210>108
<211>63
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>108
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnntccac agatttcaga 60
gca 63
<210>109
<211>48
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>109
ccttggcacc cgagaattcc annnnnnnnn nnnnnnngac cgggggta 48
<210>110
<211>58
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>110
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnnagagg tgttggtt 58
<210>111
<211>55
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>111
ccttggcacc cgagaattcc annnnnnnnn nnnnnnnacc ccctctagag cccac 55
<210>112
<211>59
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>112
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnngcttt ggtgaggga 59
<210>113
<211>62
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>113
ccttggcacc cgagaattcc annnnnnnnn nnnnnnnacc caactaaaaa tattaaacac 60
aa 62
<210>114
<211>57
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>114
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnntggag gtgggga 57
<210>115
<211>54
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>115
ccttggcacc cgagaattcc annnnnnnnn nnnnnnntca ttctatttcc ccct 54
<210>116
<211>58
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>116
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnngcagg agtccgag 58
<210>117
<211>60
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>117
ccttggcacc cgagaattcc annnnnnnnn nnnnnnntcc ttaatcattt ttattgccac 60
<210>118
<211>58
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>118
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnntgggg ataagggg 58
<210>119
<211>50
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>119
ccttggcacc cgagaattcc annnnnnnnn nnnnnnnaca aggcacacct 50
<210>120
<211>61
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>120
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnngtgta gagggaaggt 60
t 61
<210>121
<211>56
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>121
ccttggcacc cgagaattcc annnnnnnnn nnnnnnnacc ctagcaatat caacca 56
<210>122
<211>59
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>122
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnnggtca ttaggaggg 59
<210>123
<211>48
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>123
ccttggcacc cgagaattcc annnnnnnnn nnnnnnnacc cagcccat 48
<210>124
<211>61
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>124
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnntcccg tatcgaaggc 60
c 61
<210>125
<211>51
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>125
ccttggcacc cgagaattcc annnnnnnnn nnnnnnnacc acacaccacc t 51
<210>126
<211>62
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>126
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnntgtgt ttaggagtgg 60
ga 62
<210>127
<211>54
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>127
ccttggcacc cgagaattcc annnnnnnnn nnnnnnngcc cccaacaggc atca 54
<210>128
<211>61
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>128
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnntctga ggcttgtagg 60
a 61
<210>129
<211>59
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>129
ccttggcacc cgagaattcc annnnnnnnn nnnnnnntgc ttattacaat tttactggg 59
<210>130
<211>58
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>130
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnnggcgg atgaagca 58
<210>131
<211>53
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>131
ccttggcacc cgagaattcc annnnnnnnn nnnnnnntgg ctcaactttc ctc 53
<210>132
<211>64
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>132
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnntggaa gttaacggta 60
ctat 64
<210>133
<211>54
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>133
ccttggcacc cgagaattcc annnnnnnnn nnnnnnntga gggtcttact cttt 54
<210>134
<211>54
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>134
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnnggcgg ggga 54
<210>135
<211>53
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>135
ccttggcacc cgagaattcc annnnnnnnn nnnnnnnacc ccttacgagt gcg 53
<210>136
<211>68
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>136
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnntgatg attaataaga 60
gggatgac 68
<210>137
<211>57
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>137
ccttggcacc cgagaattcc annnnnnnnn nnnnnnngcc ctacaaacaa ctaacct 57
<210>138
<211>57
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>138
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnnatgag gggcatt 57
<210>139
<211>63
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>139
ccttggcacc cgagaattcc annnnnnnnn nnnnnnnttc gactcattaa attatgataa 60
tca 63
<210>140
<211>57
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>140
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnngggag tgggtgt 57
<210>141
<211>60
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>141
ccttggcacc cgagaattcc annnnnnnnn nnnnnnnact atcgctgttc attatagcta 60
<210>142
<211>62
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>142
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnnttgga gtaggtttag 60
gt 62
<210>143
<211>53
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>143
ccttggcacc cgagaattcc annnnnnnnn nnnnnnnggc ctagactacg tac 53
<210>144
<211>64
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>144
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnnggttg ttgttgattt 60
ggtt 64
<210>145
<211>48
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>145
ccttggcacc cgagaattcc annnnnnnnn nnnnnnnacc cacagcct 48
<210>146
<211>64
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>146
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnnttttt cgtgatagtg 60
gttc 64
<210>147
<211>50
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>147
ccttggcacc cgagaattcc annnnnnnnn nnnnnnngcc aacgccactt 50
<210>148
<211>66
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>148
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnnggtgt agaataggaa 60
gtatgt 66
<210>149
<211>54
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>149
ccttggcacc cgagaattcc annnnnnnnn nnnnnnnacc cgatgaggca acca 54
<210>150
<211>65
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>150
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnngctat tgtgtaagct 60
agtca 65
<210>151
<211>58
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>151
ccttggcacc cgagaattcc annnnnnnnn nnnnnnngcc caagaactat caaactcc 58
<210>152
<211>59
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>152
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnntttgt cagggggtt 59
<210>153
<211>51
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>153
ccttggcacc cgagaattcc annnnnnnnn nnnnnnnacg cctcacactc a 51
<210>154
<211>61
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>154
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnntggct gttactacga 60
g 61
<210>155
<211>53
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>155
ccttggcacc cgagaattcc annnnnnnnn nnnnnnngct cattgcatac tct 53
<210>156
<211>59
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>156
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnnactgt gagtgcgtt 59
<210>157
<211>57
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>157
ccttggcacc cgagaattcc annnnnnnnn nnnnnnntcc tcattactat tctgcct 57
<210>158
<211>62
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>158
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnnactag cacagagagt 60
tc 62
<210>159
<211>51
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>159
ccttggcacc cgagaattcc annnnnnnnn nnnnnnngcc ttacccccca c 51
<210>160
<211>60
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>160
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnngtgtg aatgagggtt 60
<210>161
<211>51
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>161
ccttggcacc cgagaattcc annnnnnnnn nnnnnnntca ctcacccacc a 51
<210>162
<211>61
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>162
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnngcctc tgttgtcaga 60
t 61
<210>163
<211>60
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>163
ccttggcacc cgagaattcc annnnnnnnn nnnnnnngtt ttcctcttgt aaatatagtt 60
<210>164
<211>62
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>164
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnnaccaa aatttttggg 60
gc 62
<210>165
<211>54
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>165
ccttggcacc cgagaattcc annnnnnnnn nnnnnnnaca gctatccatt ggtc 54
<210>166
<211>59
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>166
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnnggatg cgacaatgg 59
<210>167
<211>51
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>167
ccttggcacc cgagaattcc annnnnnnnn nnnnnnnact cataccccca t 51
<210>168
<211>69
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>168
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnngatga atattatgga 60
gaagtagtc 69
<210>169
<211>51
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>169
ccttggcacc cgagaattcc annnnnnnnn nnnnnnnacc cagctctccc t 51
<210>170
<211>59
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>170
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnnggttg ttagcggta 59
<210>171
<211>62
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>171
ccttggcacc cgagaattcc annnnnnnnn nnnnnnntct actcatcttc ctaattacca 60
ta 62
<210>172
<211>59
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>172
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnntgcta aggcgagga 59
<210>173
<211>56
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>173
ccttggcacc cgagaattcc annnnnnnnn nnnnnnngca atcctataca accgta 56
<210>174
<211>60
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>174
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnnaccta attgggctga 60
<210>175
<211>51
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>175
ccttggcacc cgagaattcc annnnnnnnn nnnnnnngcc tcaccccact a 51
<210>176
<211>63
<212>DNA
<213> Artificial Synthesis (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>176
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnngctat tttctgctag 60
ggg 63
<210>177
<211>54
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>177
ccttggcacc cgagaattcc annnnnnnnn nnnnnnntct tactcatccg cttc 54
<210>178
<211>60
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>178
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnntgcta ggtgtggttg 60
<210>179
<211>57
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>179
ccttggcacc cgagaattcc annnnnnnnn nnnnnnntca taatagttac aatcggc 57
<210>180
<211>61
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>180
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnngtgag aggtatggtt 60
t 61
<210>181
<211>62
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>181
ccttggcacc cgagaattcc annnnnnnnn nnnnnnnaca agatattcga aaaataggag 60
ga 62
<210>182
<211>63
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>182
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnngagta atagataggg 60
ctc 63
<210>183
<211>56
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>183
ccttggcacc cgagaattcc annnnnnnnn nnnnnnnaca tatcatacac aaacgc 56
<210>184
<211>59
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>184
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnngaata ggcttccgg 59
<210>185
<211>53
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>185
ccttggcacc cgagaattcc annnnnnnnn nnnnnnnacc ccattaaacg cct 53
<210>186
<211>59
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>186
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnntgtgc atagtgggg 59
<210>187
<211>56
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>187
ccttggcacc cgagaattcc annnnnnnnn nnnnnnntca actacctaac caacaa 56
<210>188
<211>69
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>188
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnntgaaa ttgttttagg 60
taatagctt 69
<210>189
<211>57
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>189
ccttggcacc cgagaattcc annnnnnnnn nnnnnnntcc tcctagacct aacctga 57
<210>190
<211>64
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>190
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnntggtt gaacattgtt 60
tgtt 64
<210>191
<211>52
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>191
ccttggcacc cgagaattcc annnnnnnnn nnnnnnnacc tattcccccg ag 52
<210>192
<211>58
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>192
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnntgatg gggtggtg 58
<210>193
<211>59
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>193
ccttggcacc cgagaattcc annnnnnnnn nnnnnnntcc ttcataaatt attcagctt 59
<210>194
<211>61
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a,c, g, or t
<400>194
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnngggaa tgatggttgt 60
c 61
<210>195
<211>52
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>195
ccttggcacc cgagaattcc annnnnnnnn nnnnnnnact cctcaatagc ca 52
<210>196
<211>62
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>196
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnnggttt agtaatgggg 60
tt 62
<210>197
<211>59
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>197
ccttggcacc cgagaattcc annnnnnnnn nnnnnnnacc cccataaata ggagaaggc 59
<210>198
<211>57
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>198
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnngggga ggtcgat 57
<210>199
<211>51
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>199
ccttggcacc cgagaattcc annnnnnnnn nnnnnnnacc ccaatacgca a 51
<210>200
<211>57
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>200
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnngcggt tgaggcg 57
<210>201
<211>54
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>201
ccttggcacc cgagaattcc annnnnnnnn nnnnnnngac tattcctagc catg 54
<210>202
<211>63
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>202
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnntccgt aatataggcc 60
tcg 63
<210>203
<211>59
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>203
ccttggcacc cgagaattcc annnnnnnnn nnnnnnngcc tcaatattct ttatctgcc 59
<210>204
<211>59
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>204
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnntgtat gggatggcg 59
<210>205
<211>57
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>205
ccttggcacc cgagaattcc annnnnnnnn nnnnnnngcc acagtaatta caaactt 57
<210>206
<211>62
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>206
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnntgcaa gaataggagg 60
tg 62
<210>207
<211>57
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>207
ccttggcacc cgagaattcc annnnnnnnn nnnnnnnttt acctttcact tcatctt 57
<210>208
<211>68
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>208
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnntgtta atgtcattaa 60
ggagagaa 68
<210>209
<211>51
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>209
ccttggcacc cgagaattcc annnnnnnnn nnnnnnnacg ccctcggctt a 51
<210>210
<211>58
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>210
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnngcaag gacgcctc 58
<210>211
<211>51
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>211
ccttggcacc cgagaattcc annnnnnnnn nnnnnnnttc tccgatccgt c 51
<210>212
<211>59
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>212
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnntggtt gtcctccga 59
<210>213
<211>49
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>213
ccttggcacc cgagaattcc annnnnnnnn nnnnnnngcc gcagacctc 49
<210>214
<211>63
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>214
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnntctga tttgtccttg 60
gaa 63
<210>215
<211>54
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>215
ccttggcacc cgagaattcc annnnnnnnn nnnnnnnttg taaaccggag atga 54
<210>216
<211>63
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>216
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnnacgaa atacatagcg 60
gtt 63
<210>217
<211>52
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>217
ccttggcacc cgagaattcc annnnnnnnn nnnnnnngaa gcagatttgg gt 52
<210>218
<211>64
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>218
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnngttga tgtgtgatag 60
ttga 64
<210>219
<211>55
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>219
ccttggcacc cgagaattcc annnnnnnnn nnnnnnngca agtacagcaa tcaac 55
<210>220
<211>61
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>220
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnnttgat ttcacggagg 60
a 61
<210>221
<211>53
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(22)..(37)
<223>n is a, c, g, or t
<400>221
ccttggcacc cgagaattcc annnnnnnnn nnnnnnngtc aaatcccttc tcg 53
<210>222
<211>57
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(30)..(45)
<223>n is a, c, g, or t
<400>222
cacgttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnnggaac gtgtggg 57
Claims (9)
1. A group of primers for human mitochondrial whole genome detection is characterized by comprising a universal primer and a specific primer.
2. The primer of claim 1, wherein the sequence of the universal primer is shown as SEQ ID No.1 and SEQ ID No. 2.
3. The primer of claim 1, wherein the specific primer sequence is shown in SEQ ID No. 3-222.
4. The specific primer of claim 3, wherein the specific primer can be modified by any one of 2-fluoro ribonucleic acid modification, 2' -O-methyl ribonucleic acid modification, 5-methyl deoxycytidine modification, deoxyinosine modification, 2-aminopurine modification, 5-bromo-deoxyuracil modification, reverse dT modification, dideoxycytidine modification, spacer modification, amino modification, thiol modification, phosphorylation modification, biotin modification, locked nucleotide modification and thio modification, or can be modified by multiple ways simultaneously.
5. A kit for detecting a mitochondrial whole genome, comprising the primer of claim 1.
6. The kit for detecting the whole mitochondrial genome is characterized by further comprising a PCR mixed reaction solution, purified magnetic beads and a positive control.
7. The kit of claim 6, wherein the PCR mix reaction solution comprises DNA polymerase, dNTPs and 2 XPCR buffer.
8. A method of detecting a mitochondrial whole genome of non-diagnostic interest, the method comprising the steps of: combining the specific primer described in patent claim 3 with the template genome DNA sequence, amplifying the target region of the sample to be detected by using the general primer, purifying the library by using magnetic beads, carrying out high-throughput sequencing on the obtained library and analyzing the mutation of mitochondrial genes.
9. The method of claim 8, wherein the PCR reaction conditions are:
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| CN201911286581.9A CN110885880A (en) | 2019-12-14 | 2019-12-14 | Primer, method and kit for human mitochondria whole genome detection |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111676280A (en) * | 2020-07-29 | 2020-09-18 | 中国人民解放军军事科学院军事医学研究院 | Primer combination for mitochondrial hypervariable region composite amplification based on high-throughput sequencing technology, sequencing method and application of primer combination |
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| CN104480207A (en) * | 2014-12-17 | 2015-04-01 | 杭州吉洛生物医药科技有限公司 | Primer pair capable of detecting specificity of human mitochondrial genome |
| CN106929582A (en) * | 2017-03-21 | 2017-07-07 | 杭州联川基因诊断技术有限公司 | A kind of method and kit for detecting EGFR genetic mutation |
| CN107988372A (en) * | 2018-01-03 | 2018-05-04 | 杭州联川基因诊断技术有限公司 | A kind of kit and its detection method for detecting susceptibility gene of colorectal cancer mutation |
| CN109402257A (en) * | 2018-11-03 | 2019-03-01 | 杭州链康医学检验实验室有限公司 | A kind of primer, method and kit for the detection of the gene whole coding sequence mutational site mankind BRCA1 and BRCA2 |
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2019
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104480207A (en) * | 2014-12-17 | 2015-04-01 | 杭州吉洛生物医药科技有限公司 | Primer pair capable of detecting specificity of human mitochondrial genome |
| CN106929582A (en) * | 2017-03-21 | 2017-07-07 | 杭州联川基因诊断技术有限公司 | A kind of method and kit for detecting EGFR genetic mutation |
| CN107988372A (en) * | 2018-01-03 | 2018-05-04 | 杭州联川基因诊断技术有限公司 | A kind of kit and its detection method for detecting susceptibility gene of colorectal cancer mutation |
| CN109402257A (en) * | 2018-11-03 | 2019-03-01 | 杭州链康医学检验实验室有限公司 | A kind of primer, method and kit for the detection of the gene whole coding sequence mutational site mankind BRCA1 and BRCA2 |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111676280A (en) * | 2020-07-29 | 2020-09-18 | 中国人民解放军军事科学院军事医学研究院 | Primer combination for mitochondrial hypervariable region composite amplification based on high-throughput sequencing technology, sequencing method and application of primer combination |
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